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Abstract

Background

Macroautophagy is an evolutionarily conserved mechanism for bulk intracellular degradation
of proteins and organelles. Pathological studies have implicated macroautophagy defects
in human neurodegenerative disorders of aging including Alzheimer’s disease and tauopathies.
Neuronal deficiency of macroautophagy throughout mouse embryonic development results
in neurodevelopmental defects and early postnatal mortality. However, the role of
macroautophagy in mature CNS neurons, and the relationship with human disease neuropathology,
remains unclear. Here we describe mice deficient in an essential macroautophagy component,
Atg7, specifically within postnatal CNS neurons.

Conclusions

These data elucidate a role for macroautophagy in the long-term survival and physiological
function of adult CNS neurons. Neurodegeneration in the context of macroautophagy
deficiency is mediated through a phospho-tau pathway.

Background

The primary etiologies of neurodegenerative disorders, including Alzheimer’s disease
(AD), frontotemporal dementia (FTD) and Parkinson’s disease (PD), remain largely unknown,
but common pathological features suggest a role for altered protein degradation. For
instance, proteinaceous intracellular inclusions composed in part of aggregated α-synuclein
protein, termed Lewy bodies, typify PD brain pathology, whereas neurofibrillary tangles
(NFT) and Pick bodies containing phosphorylated tau protein are commonly found in
the context of taupathies such as AD and FTD. Rare, inherited familial forms of neurodegenerative
diseases
[1] are caused by mutations in genes encoding these accumulated proteins, such as α-synuclein
[2,3] in PD and tau in FTD, but the vast majority of patients do not harbor known mutations.
Thus, it has been hypothesized that in these ‘sporadic’ cases, pathological inclusions
may reflect broadly defective protein degradation through mechanisms such as the ubiquitin-proteasome
system (UPS)
[4] and macroautophagy
[5,6]. The latter is of particular interest because of its apparent role in the degradation
of protein aggregates and inclusions
[7].

Macroautophagy is a pathway of bulk cytoplasmic protein and organelle degradation
characterized by double-membrane vesicles that engulf cargo and target it to lysosomes
for degradation
[8]. The pathway is typically induced in the context of starvation or other stressors.
Defects in the macroautophagy process may theoretically occur at a variety of steps,
from the initial formation of a pre-autophagosome limiting membrane, to the ultimate
fusion of mature autophagosomes with the lysosomal compartment
[9]. Macroautophagy defects have been well described on pathological analyses of brain
sections from patients with a variety of neurodegenerative disorders, including AD,
PD and FTD
[5,10]. Furthermore, inherited genetic forms of neurodegeneration are associated with mutations
in the macroautophagy-lysosomal pathway
[11,12]. Finally, as macroautophagy dysfunction is a well-documented feature of aging, it
has been implicated in the age-dependent nature of the major neurodegenerative disorders
[5,9,10].

Genetically altered mice that are deficient in essential macroautophagy pathway components,
Atg5 or Atg7, throughout neural development, display reduced neuronal survival and
harbor ubiquitin-positive inclusions in the cell soma
[13-16]. But surprisingly, prevention of inclusion formation in the context of Atg7-deficiency
by a second genetic ablation of p62, which encodes an ubiquitin-binding protein associated
with autophagosomes, does not suppress neurodegeneration, arguing against a toxic
role for inclusions
[17]. Thus, the mechanism of neuronal loss with macroautophagy deficiency, and how this
relates to neurodegeneration, remains unclear.

Here we generated conditional Atg7-deficient mice specifically within mature CNS neurons.
Atg7-deficient neurons were defective in the initiation of macroautophagy, and displayed
a progressive degeneration with prominent inclusions that harbor ubiquitin, p62, phosphorylated
tau and GSK3β. The mutant mice exhibited behavioral deficits consistent with the pathological
changes. Furthermore, pharmacological or genetic suppression of tau phosphorylation
effectively inhibited neurodegeneration in the context of Atg7-deficiency in vivo.

Furthermore, numerous ubiquitin-positive inclusions were apparent in essentially all
Atg7-deficient CA1 cell bodies from 2-month of age, whereas these were never seen
in the control CamK-Atg7 cWT mice (Figure
1e). These inclusions were stained positive for p62
[17,21], which is a component of the macroautophagy machinery pathway (Additional file
1), and further confirmed the macroautophagy defect in forebrain neurons. In contrast,
such inclusions were absent from the CA3 neurons (data not shown). Further analysis
by electron microscopy revealed that these inclusions were composed of both filamentous
and vesicular elements (Figure
1f).

We further compared CamK-Atg7 cKO neurodegeneration with the effect of Atg7 deficiency in a second population of
mature CNS neurons, midbrain dopamine (DA) neurons. To this end, we generated animals
that express CRE under the control of the dopamine transporter (Dat) gene regulatory
elements, and are homozygous for the floxed Atg7 allele (DatCre/+Atg7flox/flox; Dat-Atg7 cKO mice rather than CamK-Atg7 cKO mice). Dat-Atg7 cKO mice displayed a very similar pathological progression to CamK-Atg7 cKO mice with cytoplasmic ubiquitin- and p62-positive inclusions, albeit the process
is selective for midbrain DA neurons as expected (Additional file
2c,d). Neurodegeneration progresses appeared more rapid in the Dat-Atg7 cKO mouse model than the CamK-Atg7 cKO mouse model (25% midbrain DA neuron lost at 2-months of age and 38% lost at 4-month;
Additional file
2a,b).

We further examined the physiological and behavioral consequences of Atg7-deficiency
within forebrain neurons. Extracellular recording of field potentials were performed
at Schaffer collateral synapses in area CA1 of acutely prepared hippocampal slices
from 3-month-old male CamK-Atg7 cKO mice and control CamK-Atg7 cWT littermates. CamK-Atg7 cKO mice showed normal input/output amplitudes in response to single stimuli (Figure
2a), as well as intact paired-pulse facilitation (PPF) at a variety of interpulse intervals
(Figure
2b). These findings suggest that there are no gross differences in synaptic organization
or baseline synaptic transmission in the cKO mice at this age. In contrast, early
long-term potentiation (E-LTP) induced by a single high-frequency tetanic stimulation
- a long-lasting protein synthesis-independent form of synaptic potentiation - was
impaired in CamK-Atg7 cKO slices (Figure
2c). In contrast, we note that long-term depression was intact in the cKO mice (data
not shown). The relatively selective physiological impairment is unlikely to be secondary
to the limited cell loss.

Phospho-tau-positive inclusions in Atg7-deficient neurons

We investigated whether neurodegeneration caused by Atg7-deficiency is associated
with typical pathological hallmarks of human neurodegenerative syndromes. Macroautophagy
has previously been implicated in the clearance of various proteins implicated in
human neurodegenerative syndromes including Alzheimer precursor protein (APP), α-synuclein,
TDP-43, tau, and huntingtin
[22-29]. However, direct in vivo evidence of an essential role for macroautophagy in the degradation of these proteins
in forebrain is lacking. No accumulation of APP (or the APP-derived peptide fragmant
β-amyloid), α-synuclein, or TDP-43 was detected in CamK-Atg7 cKO mouse brain (Additional file
3a, b). However, cytoplasmic inclusions in Atg7-deficient CA1 pyramidal neurons and
cerebral cortex neurons were prominently stained with multiple well-characterized
antibodies to phospho-tau including AT8 (epitope at Ser202/Thr205), AT100 (epitope
at Ser212/Thr214), and TG3 (epitope at Thr231/Ser235)
[30,31] (Figure
3a-c). Similarly, electron microscopic analysis confirmed TG3-positive staining in
the cytoplasmic inclusions of Atg7-deficient neurons (Figure
3d). We note that the inclusions were not stained with other antibodies for mature
phospho-tau positive inclusions in human pathology, AT270 (epitope at Ser181) and
PHF1 (epitope at Ser396/Ser404). Furthermore, the cytoplasmic inclusions did not stain
with Thioflavin S, which marks mature NFTs in human tauopathies (Additional file
3c).

Quantitative Western blotting of forebrain extracts revealed that phospho-tau protein
epitopes were broadly increased in forebrain tissues from CamK-Atg7 cKO mice, whereas total tau protein appeared unaltered (Figure
3e). Several epitopes, including AT8, AT100, and TG3, were increased in both 0.5% TritinX-100-soluble
and insoluble brain extracts (relative to CamK-Atg7 cWT controls; Figure
3e), whereas AT180 accumulated only in insoluble extracts, and accumulation was not
altered for AT270 and PHF1 (Figure
3e). The phospho-tau epitope staining pattern appeared very similar in midbrain DA
neurons of Dat-Atg7 cKO mice (Additional file
2e, Figure
4e). A similar phospho-tau pattern has previously been suggested to represent an early
‘pre-tangle’ state
[32]; this may reflect an early stage of non-fibrillar tau aggregation prior to its assembly
into paired helical filaments (PHF). Taken together, these data implicate phospho-tau
accumulation in Atg7-deficiency-mediated neurodegeneration. However, the phospho-tau
aggregates in the context of Atg7-deficient neurons do not replicate aspects of mature
human tauopathy pathology.

GSK3β staining at phospho-tau inclusions in Atg7-deficient neurons

Given the accumulation of phosphorylated -- but not total -- tau in Atg7-deficient
neurons (Figure
4e), we hypothesized that a kinase that is known to phosphorylate tau, such as GSK3β,
may be altered. Immunostaining of cortical neurons revealed dramatic re-localization
of GSK3β, including both active (epitope at Tyr216) and inactive (epitope at Ser9)
phosphorylated forms, to phospho-tau-positive and ubiquitin/p62-positive inclusions
in Atg7-deficient neurons (Figure
4a-c). Western blot analysis confirmed that total and phosphorylated forms of GSK3α/β
were increased in forebrain tissue extracts from CamK-Atg7 cKO mice, compared to CamK-Atg7 cWT mice (Figure
4d). Another kinase implicated in phosphorylation of tau, CDK5, did not appear to be
re-localized to the inclusions in Atg7-deficient neurons
[33] (Additional file
4d). Inclusions in Atg7-deficient neurons stained positively for a second microtubule-associated
GSK3β substrate, phospho-CRMP2
[34] (Additional file
4a,b). In contrast, β-Catenin, a well-described GSK3β substrate in the context of Wnt
signaling pathway, did not appear altered in staining in Atg7-deficient neurons (Additional
file
4c). Thus, accumulated GSK3β in the context of Atg7-deficiency appears to display substrate
specificity, perhaps related to subcellular re-localization at inclusions.

Pharmacological or genetic inhibition of phospho-tau accumulation can rescue neuronal
cell death in vivo

To examine the causality between phospho-tau and neurodegeneration in the context
of Atg7-deficiency, we sought to determine whether neurons deficient in Atg7 could
be effectively protected in vivo through the modulation of phospho-tau production. We focused these ‘rescue’ studies
on Dat-Atg7 cKO mice (rather than CamK-Atg7 cKO mice) because the neurodegeneration progresses more rapidly in Dat-Atg7 cKO mouse model than CamK-Atg7 cKO mouse model, as noted above, and the degenerative and pathological processes
are restricted to a single cell type in the Dat-Atg7 cKO mice (midbrain DA neurons; Additional file
2a,b). Dat-Atg7 cKO mice also displayed a very similar pathological progression to CamK-Atg7 cKO mice with cytoplasmic ubiquitin- and p62-positive inclusions (Additional file
2c,d) that further stain for phospho-tau and GSK3β (Additional file
2e,f). Thus, analysis of pathology in Dat-Atg7 cKO mice affords a more facile and accurate quantification of the cell autonomous
impact of macroautophagy on the loss of mature CNS neurons.

To investigate the role of phospho-tau accumulation in Atg7-deficiency-induced neurodegeneration,
Dat-Atg7 cKO or Dat-Atg7 cWT mice were treated chronically with a potent GSK3β/CDK5 inhibitor, Alsterpaullone
(5 mg/kg/d, i.p.) for a period of 3 weeks starting at 5-week of age
[35]. Alsterpaullone can inhibit the activities of GSK3β, as well as several other tau
kinases (CDK1/2/5, GSK3α, and, to lesser extent, ERK1/2 and PKA) to suppress tau phosphorylation
(Additional file
5a)
[36]. At the end of the treatment course (8-weeks of age), pathological examination of
the mice revealed that Alsterpaullone treatment led to a significant increase in the
survival of midbrain DA neurons in Dat-Atg7 cKO mice (24.3% increased survival, p < 0.01), whereas Alsterpaullone-treated control
Dat-Atg7 cWT mice appeared unaltered (Figure
5a, b). In contrast, ubiquitin-positive inclusions were unchanged in size and number
in Alsterpaullone-treated Dat-Atg7 cKO mice, whereas no inclusions were seen in Alsterpaullone-treated Dat-Atg7 cWT mice (Additional file
5b, c). This is consistent with the previous report that the inclusion formation and
neurodegeneration are independent in the context of macroautophagy deficiency
[17]. These in vivo results are suggesting a protective effect by phospho-tau inhibition in the context
of macroautophagy deficiency-induced neurodegeneration. As Alsterpaullone does display
some inhibitory activity at kinases in addition to GSK3β, such as CDK5
[36], we cannot exclude additional in vivo kinase targets. But we note that unlike GSK3β, CDK5 did not appear modified or re-localized
in Dat-Atg7 cKO neurons (Additional file
4d).

Next, we examined the effect of tau-deficiency
[37] in Dat-Atg7 cKO mice. We generated Dat-Atg7/tau double cKO (DatCre/+Atg7flox/floxtau-/-) mice, and compared the loss of midbrain DA neuron in Dat-Atg7 single cKO (DatCre/+Atg7flox/floxtau+/+ or DatCre/+Atg7flox/floxtau+/-) and Dat-Atg7/tau double cKO mice. The loss of midbrain DA neurons in Dat-Atg7 cKO mice was significantly rescued in Dat-Atg7/tau double cKO mice at the age of 3-month (Figure
5c,d). Again, the formation of ubiquitin-positive inclusion was not changed in Dat-Atg7/tau double cKO mice (Additional file
5d,e). Consistent with the previous report that tau-deficiency alone led to no abnormality
in the brain
[37,38], neither neurodegeneration nor ubiquitin/p62-positive inclusions was seen in the
midbrain DA neurons of tau KO mice (Figure
5c,d and Additional file
5d,e). Taken together, these approaches support a model whereby accumulation of phospho-tau
contributes to neurodegeneration in the context of macroautophagy-deficiency, whereas
the formation of ubiquitin/p62-positive inclusions is independent of phospho-tau signaling.

Discussion

Here we investigated mechanisms of neurodegeneration downstream of Atg7-deficiency,
and describe the pathological accumulation of GSKβ and phospho-tau proteins. A striking
feature of neuropathology in the context of Atg7-deficiency is the redistribution
of GSK3β to inclusions. We note that both GSK3β and phospho-tau are reported to be
found in inclusions in tauopathy patient brain
[39-43]. However, it is important to emphasize that Atg7-deficiency does not appear to induce
a full tauopathy pathology, as not all phospho-tau epitopes are observed (e.g., PHF1
antibody is negative, Figure
4e), and amyloid staining with Thioflavin S, as well as electron microscopic analysis,
do not support the presence of mature NFTs. A similar phospho-tau pattern has previously
been suggested to represent an early ‘pre-tangle’ pathological state
[32], thought to reflect non-fibrillar tau aggregation prior to assembly into PHFs. Such
non-fibrillar hyperphosphorylated tau, rather than mature NFTs, may be the relevant
toxic form in vivo in the context of neurodegeneration and behavioral impairment
[44]. Hoozemans et al. reported phospho-tau-positive pre-tangles with accumulation of GSK3β, ubiquitin and
p62 in postmortem specimens of AD patients
[45], reminiscent of pathology in Atg7-deficient neurons in vivo. Phospho-tau pathology as seen in Atg7-deficient animals may broadly relate to neuronal
dysfunction in neurodegeneration, as macroautophagy deficiency and phospho-tau are
commonly observed in a broad array of neurodegenerative disorders including AD, PD,
tauopathy, huntington disease, amyotrophic lateral sclerosis, and Gaucher disease
[6,46-49]. Although genetic mutations in ATG7 have not been described in human disease, mutations within other components of the
macroautophagy-lysosomal pathway underlie tauopathies
[50], consistent with our observations in the mouse model.

The in vivo pharmacological and genetic ‘rescue’ studies herein suggest a role for phospho-tau
accumulation in neurodegeneration downstream of Atg7-deficiency. In contrast, prior
attempts to rescue macroautophagy-deficiency associated neurodegeneration by preventing
the formation of aggregates, by generation of double-knockout mice deficient in Atg7
as well as p62, were unsuccessful
[17], suggesting that inclusion formation per se is insufficient for degeneration. It is interesting to note that nonetheless, p62
deletion does rescue the Atg7 deficiency-associated cell loss in hepatocytes
[17], and thus degenerative pathways downstream of macroautophagy loss appear cell type-specific.
Furthermore, within the CNS, various neuronal subtypes appear to be differentially
affected by macroautophagy deficiency. Purkinje neurons deficient in Atg7 display
axonal swellings and are rapidly lost
[51]. TH-positive midbrain DA neurons display axonal dystrophy and degeneration, ubiquitin/p62-positive
inclusions, and delayed cell loss and locomotor dysfunction
[52]. Although tau pathology was not investigated in these other models, staining for
the Parkinson’s disease associated proteins α-synuclein and leucine rich repeat kinase-2
(LRRK2) was reported in Atg7-deficient DA neurons
[52]. We failed to detect evidence of α-synuclein accumulation in our analysis of either
midbrain DA neuron-selective or forebrain neuron-selective Atg7-deficient mice detailed
above (data not shown). Such discrepancies may reflect differences in the selectivity
or timing of the CRE-mediated deletion strains used in the different studies, or selective
sensitivity to macroautophagy loss across distinct neuron types. We note that phospho-tau
pathology was apparent in the context of either midbrain DA neuron-selective or forebrain
neuron-selective Atg7-deficiency.

The molecular basis of GSK3β and phospho-tau accumulation in Atg7-deficient neurons
remains to be elucidated. We cannot exclude the possibility that GSK3β accumulation
is a secondary effect of phospho-tau accumulation. A recent study described intracellular
redistribution of GSK3β to multivesicular bodies, albeit in the context of Wnt pathway
modulation
[53]. As multivesicular bodies directly associate with the macroautophagy machinery, it
is possible that GSK3β degradation is selectively modified with macroautophagy loss
[54]. Although GSK3β is a strong candidate for the relevant upstream kinase, we hypothesize
the involvement of other kinase pathways, particularly given the multiple targets
of the pharmacological kinase inhibitor used, Alsterpaullone. Furthermore, Alsterpaullone-mediated
protection may be mediated through targets in addition to tau, which would be of further
interest.

We propose a role for basal macroautophagy in regulating the metabolism of phospho-tau
proteins at physiological or pre-pathological state (Figure
5e). In the context of macroautophagy loss, GSK3β and phospho-tau are accumulated,
reminiscent of early pathology that precedes human tauopathy. It is interesting to
note that both GSK3β and tau are believed to be potent upstream regulators of macroautophagy
[55-58]. We hypothesize that this may reflect a feedback loop, where defective macroautophagy
leads progressively to more accumulation of phospho-tau and GSK3β, and in turn the
accumulated phospho-tau and GSK3β both induce macroautophagy activity. Initially such
feedback may be effective, although the accumulated proteins form inclusions. But
once macroautophagy deficiency is complete, as in late-stage disease or in knockout
mice, this feedback would be ineffective. Thus, such a feedback circuit may be an
important pathway to rejuvenate the macroautophagy pathway, which is known to wane
with aging
[59].

Conclusions

Atg7 cKO in mouse forebrain neurons led to an age-dependent neurodegeneration with ubiquitin/p62-positive
and phospho-tau/GSK3β inclusions, but not the full pathological features of NFTs in
tauopathy. Pharmacological or genetic inhibition of tau phosphorylation in vivo successfully rescued neurodegeneration in the context of macroautophagy-deficiency.
As GSK3β and tau are also upstream inducers of macroautophagy, this implicates a negative
feedback loop in human pathology.

Neuron counting

To obtain neuronal cell count, 50 μm coronal brain sections were made using a vibratome.
In order to count CA1 neurons, the first 30 sections from the rostral hippocampus
were stained with rabbit anti-MAP2 antibody (AB5622, Millipore) at a dilution of 1:500,
as well as NeuroTraceTM Fluorescent Nissl stain (N21480, Invitrogen). MAP2-positive neurons were visualized
using a Cy3-conjugated secondary antibody (Jackson ImmunoResearch). MAP2 and Nissl
double-positive neurons in the CA1 regions were counted manually. In order to count
TH-positive neurons, sections covering the entire substantia nigra (25-30 sections
/ mouse) were stained with sheep anti-TH antibody (P60101, Pel-Freez) at a dilution
of 1:250. TH-positive neurons were visualized using the ABC Kit (PK6106, Vector Laboratories)
and DAB (SK4100, Vector Laboratories). TH-positive neurons in the substantia nigral
regions were counted manually under the light microscope.

Electron microscopy

Electron microscopic analysis was as described
[61]. Anesthetized mice were perfused and fixed in PBS containing 4% paraformaldehyde
and 0.5% gultaralaldehyde. The brains were post-fixed at 4°C for 2 h, and the 80 μm
vibratome sections were made. The sections were treated in 1% osmium tetroxide, then
dehydrated in pure ethanol and infiltrated overnight with Epon 812. Epon was polymerized
at 60°C for 24 h, cooled and embedded in a larger Epon capsule. Ultrathin sections
were cut with an MT5000 ultramicrotome, stained with uranyl acetate and lead citrate.
Images were taken with a JOEL 100S Electron Microscope (JOEL USA).

Tissue fractionation

Preparation of soluble and insoluble fractions was performed as described with some
modifications
[14]. Cortical and hippocampal tissues from mouse brains were homogenized in 5× volume
of ice-cold 0.25M sucrose buffer (50mM Tris-HCl [pH7.6]) containing protease inhibitors
(P8340, Sigma) and phosphatase inhibitors (#78420, Thermo Scientific). The homogenized
tissues were centrifuged at 500× g for 10 min at 4°C. The supernatants were lysed
with an equal volume of cold sucrose buffer containing 1% Triton X-100. The lysates
were centrifuged at 13,000× g for 15 min at 4°C. The supernatants contained the soluble
fraction. The pellets were resuspended in 1% SDS in PBS (insoluble fraction). Both
fractions were subjected to standard Western Blotting analysis. The antibodies used
here are: anti-phospho-tau AT8, AT100, AT180, AT270, TG3 and PHF1, anti-Tau1 and anti-Actin
(ab3280, Abcam). Horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch)
and SuperSignal West Pico or Dura (#34077, 34075, Pierce) were used for detection.

Fear conditioning

10-13-mon-old male CamK-Atg7 cWT or CamK-Atg7 cKO mice were used (n = 8 - 10). The mice were placed in a conditioning chamber (Med
Associates) for 2 min before the onset of a tone (conditioned stimulus) (30 s, 85
dB sound at 2800 Hz) and conditioned by a single electrical foot shock (0.45 mA) in
the last 2 s. The mice were left in the chamber for another 30 s and placed back into
their home cage. Contextual fear learning was measured in the same chamber 24 h after
the training by monitoring the freezing for 5 min without electrical shock. Cued fear
learning was measured 24 h after the contextual testing. The mice were placed in a
novel chamber for 2 min (pre-conditioning). After that, the mice were exposed to the
conditioned stimulus for 3 min, and the freezing was monitored. Freezing behavior
was scored using FreezeView software (Med Associates Inc.).

Drug injection

Five-week-old Dat-Atg7 cWT and Dat-Atg7 cKO mice were treated with Alsterpaullone (A1136, A.G. Scientific)
[35]. The drug was dissolved in saline containing 20% DMSO/ 25% Tween80, sonicated, and
injected intraperitoneally at a dose of 5 mg/kg every day for 3 weeks. After the final
injection, the mice were perfused and processed for histological analyses. We used
Dat-Atg7 cWT mice as controls for Dat-Atg7 cKO mice, to address potential phenotypes due to Cre transgene inserted at the DAT
locus
[62].

Statistical analysis

All comparisons between groups were made using the Mann-Whitney U-test (for two samples)
or non-repeated measures ANOVA (for multiple samples). The values are expressed as
the means ± S.E. A p value less than 0.05 is considered significant.

Competing interests

The authors declare no competing interests.

Authors’ contributions

KI, JR, HK, EC, JK, and MK performed the experiments. KI, HK, EK, EC, and AA analyzed
the results. KI and AA designed the study and wrote the manuscript. All authors read
and approved the final manuscript.

Acknowledgements

We would like to thank G. Di Paulo, and O. Hobert for suggestions and comments on
the manuscript, R. Hen for generously providing DatCre/+ mice, P. Davies for generously providing phospho-tau antibodies, E. Kominami, T.
Chiba, and K. Tanaka for generously providing Atg7flox/flox mice, J.Q. Trojanowski and D. Dickson for electron microscopic analysis, and T. Iwasato,
J. Dunning, C. Doege, H. Rhinn, D. MacLeod, W. Vanti, S. Vasishta for technical help.
This work was supported by grants from Kanae Foundation for the Promotion of Medical
Science, and Research Foundation ITSUU Laboratory to K.I. K.I. was a postdoctoral
fellow of New York Stem Cell Foundation. This work was supported by grants from the
Michael J. Fox Foundation, NINDS, and NIA to A.A.